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Properties and efficient scrap-and-build repairing of mechanically sheared 3' DNA ends.

ABSTRACT: Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3' termini (about 20% of the total) was not repaired after the removal of 3' phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3'-?>?5' exonuclease activity of exonuclease III (3'-?>?5' exonuclease, insensitive to the 3' phosphate group) was counterbalanced by the 5'-?>?3' polymerase activity of T4DP. This combined reaction, termed "SB-repairing" (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.

SUBMITTER: Ohtsubo Y

PROVIDER: S-EPMC6841706 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Properties and efficient scrap-and-build repairing of mechanically sheared 3' DNA ends.

Ohtsubo Yoshiyuki Y Sakai Keiichiro K Nagata Yuji Y Tsuda Masataka M

Communications biology 20191108

Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3' termini (about 20% ...[more]

PMID: 31728420

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